An outbreak of acute hepatitis of unknown aetiology in children was reported in Scotland1 in April 2022 and has now been identified in 35 countries2. Several recent studies have suggested an association with human adenovirus with this outbreak, a virus not commonly associated with hepatitis. Here we report a detailed case–control investigation and find an association between adeno-associated virus 2 (AAV2) infection and host genetics in disease susceptibility. Using next-generation sequencing, PCR with reverse transcription, serology and in situ hybridization, we detected recent infection with AAV2 in plasma and liver samples in 26 out of 32 (81%) cases of hepatitis compared with 5 out of 74 (7%) of samples from unaffected individuals. Furthermore, AAV2 was detected within ballooned hepatocytes alongside a prominent T cell infiltrate in liver biopsy samples. In keeping with a CD4+ T-cell-mediated immune pathology, the human leukocyte antigen (HLA) class II HLA-DRB1*04:01 allele was identified in 25 out of 27 cases (93%) compared with a background frequency of 10 out of 64 (16%; P = 5.49 × 10−12). In summary, we report an outbreak of acute paediatric hepatitis associated with AAV2 infection (most likely acquired as a co-infection with human adenovirus that is usually required as a ‘helper virus’ to support AAV2 replication) and disease susceptibility related to HLA class II status. A case–control study investigating the causes of recent cases of acute hepatitis of unknown aetiology in 32 children identifies an association between adeno-associated virus infection and host genetics in disease susceptibility.

On 31 March 2022, Public Health Scotland was alerted to five children aged 3-5 years admitted to hospital with severe hepatitis of unknown aetiology. Retrospective investigation identified eight additional cases aged 10 years and younger since 1 January 2022. Two pairs of cases have epidemiological links. Common viral hepatitis causes were excluded in those with available results. Five children were adenovirus PCR-positive. Other childhood viruses, including SARS-CoV-2, have been isolated. Investigations are ongoing, with new cases still presenting.

Background During the 2017/18 and 2018/19 influenza seasons, molecular amplification-based point-of-care tests (mPOCT) were introduced in Scotland to aid triaging respiratory patients for hospital admission, yet communication of results to national surveillance was unaccounted for. Aim This retrospective study aims to describe steps taken to capture mPOCT data and assess impact on influenza surveillance. Methods Questionnaires determined mPOCT usage in 2017/18 and 2018/19. Searches of the Electronic Communication of Surveillance in Scotland (ECOSS) database were performed and compared with information stored in laboratory information management systems. Effect of incomplete data on surveillance was determined by comparing routine against enhanced data and assessing changes in influenza activity levels determined by the moving epidemic method. Results The number of areas employing mPOCT increased over the two seasons (6/14 in 2017/18 and 8/14 in 2018/19). Analysis of a small number of areas (n = 3) showed capture of positive mPOCT results in ECOSS improved between seasons and remained high (> 94%). However, capture of negative results was incomplete. Despite small discrepancies in weekly activity assessments, routine data were able to identify trend, start, peak and end of both influenza seasons. Conclusion This study has shown an improvement in capture of data from influenza mPOCT and has highlighted issues that need to be addressed for results to be accurately captured in national surveillance. With the clear benefit to patient management we suggest careful consideration should be given to the connectivity aspects of the technology in order to ensure minimal impact on national surveillance.

Nucleic acid amplification tests (NAATs) are the most sensitive method for diagnosing chlamydia and gonorrhoea. We use the COBAS 4800 CT/NG combined assay (Roche Molecular Diagnostics, CA, USA), and whilst the majority of samples yield definitive results, a small proportion are reported as indeterminate. In these instances, it is usual practice to request repeat samples which delays diagnosis. This audit was twofold: first to establish the proportion of indeterminate results with current NAAT testing requiring re-sampling. Second, to determine whether a second NAAT such as Cepheid GeneXpert CT/NG assay (Cepheid, CA, USA) could be used on initial indeterminate samples to resolve indeterminate results, therefore reducing need for repeat sampling. During 2012, 144/21,931 (0.66%) samples were indeterminate for Neisseria gonorrhoeae, Chlamydia trachomatis or both, and a repeat sample was received in only 51.77% of patients with final results being delayed for more than 24 h. Over the next six months, there were 77/9472 (0.81%) indeterminate results. After an evaluation and introduction of the Cepheid assay, the number of indeterminate results fell to 9 (0.10%). Thus, use of the Cepheid assay significantly reduced indeterminate results, reduced reliance on a repeat sampling and significantly improved turnaround time, laboratory workflow and patient experience.

Gene sequence diversity plays an important function in determining survival of micro-organisms. Pathogenicity of HIV is correlated to host as well as viral factors. We aimed to identify sequence variations in tat, nef and the membrane-proximal gp41. These genes regulate important viral functions: tat for trans-activation, nef for enhancing infectivity and the membrane-proximal gp41 for fusion which could correlate with HIV disease progression. We studied HIV sequences from ART naive adult Ugandans. Sequence diversity was analysed for 19 rapid progressors and 22 long-term survivors, Rapid progressors were individuals who progressed to a CD4 count of 500 cells/µl after a median time of 8.8 (range 7.5 to 9.3) years, measured from the time of the index HIV sero-positive result to the time of obtaining the study blood sample. Amplification of DNA by polymerase chain reaction (PCR) and subsequent sequencing of tat, nef and membrane-proximal gp41 was performed starting from viral DNA directly obtained from frozen uncultured peripheral blood mononuclear cells. A ‘long’ tat protein was only observed in rapid progressors (RPs). The ‘long’ tatappears to predict rapid disease progression and could be relevant for designing an HIV-1 prognostic assay. Key words: HIV-1, progression, tat, nef, gp41.

In common with reports from other European countries, we describe a substantial increase in the number of laboratory reports of Mycoplasma pneumoniae in Scotland in 2010 and 2011. The highest number of reports came from those aged one year and younger. However, reports from young children were more likely to come from PCR testing than serological testing.

Background Boosted protease inhibitor (bPI) monotherapy (bPImono) potentially has substantial cost, safety and operational benefits. It has never been evaluated as second-line antiretroviral therapy (ART) in Africa. Methods After 24 weeks of lopinavir/ritonavir- containing second-line therapy, DART participants were randomized to remain on combination therapy (CT), or change to bPI-mono maintenance (SARA trial; ISRCTN53817258). Joint primary end points were CD4+ T-cell changes 24 weeks later and serious adverse events (SAEs); retrospectively assayed viral load (VL) was a secondary end point. Analyses were intention-to-treat. Results A total of 192 participants were randomized to CT ( n=95) or bPImono ( n=97) and followed for median 60 weeks (IQR 45–84). Participants received median 4.0 years (IQR 3.5–4.4) first-line ART. Median CD4+ T-cell count at first-line failure was 86 cells/mm3 (47–136), increasing to 245 cells/mm3 (173–325) after 24-week induction when 77% had VL<50 copies/ml. Overall, 44 (23%) were receiving second-line therapy with bPI and nucleoside reverse transcriptase inhibitors (NRTI) only, and 148 (77%) with bPI plus non-NRTI (NNRTI) with or without NRTI. At 24 weeks after randomization to CT versus bPImono, mean CD4+ T-cell increase was 42 (CT, n=85) versus 49 cells/mm3 (bPImono, n=88; adjusted difference 13 [95% CI -15, 43], P=0.37; non-inferior compared with predetermined non-inferiority margin [-33]). Virological suppression was greater for CT versus bPImono (trend P=0.009): 77% (70/91) versus 60% (56/94) were <50 copies/ml, and 5% (5) versus 14% (13) were ≥1,000 copies/ml, respectively. A total of 0 (0%) versus 5 (5%) participants had major protease inhibitor mutations and 3 (3%) versus 0 (0%) new NNRTI/NRTI mutations were detected during follow-up. Two participants (1 CT and 1 bPImono) died >24 weeks after randomization, and 5 (2 CT and 3 bPImono) experienced SAEs ( P=0.51). Conclusions bPImono following a 24-week second-line induction was associated with similar CD4+ T-cell response, but increased low-level viraemia, generally without protease inhibitor resistance. Longer-term trials are needed to provide definitive evidence about effectiveness in Africa.

Electrochemical genosensors have undergone an enormous development in the last decades, but only very few have achieved a quantification of target content in highly processed food samples. The detection of allergens, and particularly gluten, is challenging because legislation establishes a threshold of 20 ppm for labeling as gluten-free but most genosensors expresses the results in DNA concentration or DNA copies. This paper describes the first attempt to correlate the genosensor response and the wheat content in real samples, even in the case of highly processed food samples. A sandwich-based format, comprising a capture probe immobilized onto the screen-printed gold electrode, and a signaling probe functionalized with fluorescein isothiocyanate (FITC), both hybridizing with the target was used. The hybridization event was electrochemically monitored by adding an anti-FITC peroxidase (antiFITC-HRP) and its substrate, tetramethylbenzidine. Binary model mixtures, as a reference material, and real samples have been analyzed. DNA from food was extracted and a fragment encoding the immunodominant peptide of α2-gliadin amplified by a tailored PCR. The sensor was able to selectively detect toxic cereals for celiac patients, such as different varieties of wheat, barley, rye and oats, from non-toxic plants. As low as 0.001% (10 mg/kg) of wheat flour in an inert matrix was reliably detected, which directly compete with the current method of choice for DNA detection, the real-time PCR. A good correlation with the official immunoassay was found in highly processed food samples.

Objectives: To describe the resistance mutations selected by a first-line regimen of zidovudine/lamivudine/tenofovir in the absence of real-time viral load monitoring. Design: A substudy of 300 participants from the Development of Antiretroviral Therapy in Africa trial in Uganda and Zimbabwe, which compared managing antiretroviral therapy with and without laboratory monitoring. Methods: Stored plasma samples from selected time points were assayed retrospectively for HIV-1 RNA. The pol gene in all baseline samples and those with HIV RNA >1000 copies per milliliter at weeks 24 and 48 were sequenced. Results: The proportion with HIV RNA >1000 copies per milliliter increased from 15% at 24 weeks to 24% at 48 weeks. Eighteen of 31 (58%) genotyped samples at 24 weeks had ≥1 major nucleoside reverse transcriptase inhibitor-associated mutations compared with 41 of 47 (87%) at 48 weeks. Excluding 1 nonadherent patient, a mean of 2.0 (95% confidence interval: 1.3 to 2.8) thymidine analogue mutations (TAMs) developed between weeks 24 and 48 among 14 patients with HIV RNA >1000 copies per milliliter at both time points. K65R was detected in 8 of 63 (13%) patients and was negatively associated with number of TAMs (P = 0.01) but not viral subtype (P = 0.30). Conclusions: A high rate of acquisition of TAMs, but not of K65R, among patients with prolonged viraemia was observed. However, most patients were virologically suppressed at 48 weeks, and long-term clinical and immunological outcomes in the Development of Antiretroviral Therapy in Africa trial were favorable.

BACKGROUND We investigated virological response and the emergence of resistance in the Nevirapine or Abacavir (NORA) substudy of the Development of Antiretroviral Treatment in Africa (DART) trial. METHODS Six hundred symptomatic antiretroviral-naive human immunodeficiency virus (HIV)-infected adults (CD4 cell count, <200 cells/mm(3)) from 2 Ugandan centers were randomized to receive zidovudine-lamivudine plus abacavir or nevirapine. Virology was performed retrospectively on stored plasma samples at selected time points. In patients with HIV RNA levels >1000 copies/mL, the residual activity of therapy was calculated as the reduction in HIV RNA level, compared with baseline. RESULTS Overall, HIV RNA levels were lower in the nevirapine group than in the abacavir group at 24 and 48 weeks (P < .001), although no differences were observed at weeks 4 and 12. Virological responses were similar in the 2 treatment groups for baseline HIV RNA level <100,000 copies/mL. The mean residual activity at week 48 was higher for abacavir in the presence of the typically observed resistance pattern of thymidine analogue mutations (TAMs) and M184V (1.47 log(10) copies/mL) than for nevirapine with M184V and nonnucleoside reverse-transcriptase inhibitor mutations, whether accompanied by TAMs (0.96 log(10) copies/mL) or not (1.18 log(10) copies/mL). CONCLUSIONS There was more extensive genotypic resistance in both treatment groups than is generally seen in resource-rich settings. However, significant residual activity was observed among patients with virological failure, particularly those receiving zidovudine-lamivudine plus abacavir.

To the Editors: Intermittent antiretroviral therapy strategies have been demonstrated to be suboptimal to continuous therapy.1 Nevertheless, treatment interruption (due to toxicity, problems with drug supply, or as an agent to prevent mother-to-child transmission) remains a reality for many patients. It is therefore important to study its likely impact, one important aspect of which is the emergence of drug resistance. For instance, the long half-life of nonnucleoside reverse transcriptase inhibitors encourages the selection of pre-existing resistant mutants when stopping regimens containing this class of drug, a process associated with a higher incidence of nonnucleoside reverse transcriptase inhibitor resistance.2 Tenofovir (TDF) is another drug with an extended half life, and, is increasingly being used in developing and developed world settings. The critical mutation associated with TDF resistance is K65R, and we have assessed the risk of its emergence after repeated structured treatment interruptions (STIs) with a TDF-containing regimen in the Development of Antiretroviral Therapy in Africa (DART) trial. DART is a randomized study comparing monitoring strategies in Uganda and Zimbabwe in the absence of "real-time" virological testing. In a STI substudy, 813 patients with CD4 counts ≥300 cells per microliter at 48 or 72 weeks underwent randomization 4 weeks later to either repeated cycles of 12 weeks off/on treatment or continuous therapy.3 Most patients (547) received zidovudine/lamivudine (as combivir) plus TDF. However, due to adverse clinical events, the STI substudy was stopped such that few participants had undergone multiple STI cycles.3 Because the estimated intracellular half-life of Tenofovir diphosphate (approximately 95-150 hours)4,5 is so much more longer than that for zidovudine triphosphate (9 hours) or lamivudine triphosphate (17 hours),6 we hypothesize that treatment interruption will effectively lead to Tenofovir monotherapy, with selective pressure for emergence of K65R. We examined 18 participants in the STI arm who both underwent the maximum number of complete 12-week STI cycles (4) before the substudy was closed-by definition, all these participants were randomized at week 52-and initially received combivir/TDF and remained on this regimen throughout [allowing substitution of stavudine for zidovudine (n = 2). CD4 count and viral load (HIV-1 RNA)] were measured 8 weeks into each off/on cycle (at weeks: 48, 60, 72, 84, 96, 108, 120, 132, and 144). Population sequencing and minority species assay for the mutations K65R and M184V, which are associated with resistance to TDF and lamivudine, respectively, were undertaken wherever VL >1000 copies per milliliter. For population genotypic analyses, plasma-derived HIV-1 RNA was reverse-transcribed using one-step reverse transcriptase-polymerase chain reaction (Qiagen; West Sussex, United Kingdom). cDNA encoding the entire protease gene and codons 1-320 of reverse transcriptase (RT) were amplified by nested polymerase chain reaction and sequenced using an Applied Biosystems capillary sequencer. K65R and M184V minority sequencing was performed by pyrosequencing, using PyroGold SQA sample preparation kits and a PSQ96MA analyzer (Biotage, Uppsala, Sweden). cDNA generated by reverse transcriptase-polymerase chain reaction was polymerase chain reaction-amplified using subtype-specific, allele-specific primers. Pyrosequencing was found to reproducibly detect down to mixtures of 2% mutant: 98% wild-type, consistent with previously described assays in the literature.7 At 48 weeks, that is, 4 weeks before the first STI cycle, the median (interquartile range) CD4 count was 394 (321-443) cells per cubic millimeter and all patients had a viral load <400 HIV-1 RNA copies per milliliter (14 of 18 participants had <50 HIV-1 RNA copies/mL). Viral load and CD4 plots for individual participants followed the expected patterns of CD4 cell decline and viral rebound associated with an STI strategy (representative participant shown in Fig. 1A). The level of viral rebound and resuppression was similar across successive STIs. Mean HIV-1 RNA ranged from 4.5 to 4.7 log10 copies per milliliter off treatment and declined to 2.3-2.6 log 10 copies per milliliter when treatment was resumed.FIGURE 1: Viral load and CD4 count profiles (A) for a representative participant (B), the only participant who developed resistance (M184V).M184V was first detected during the third on-treatment cycle in 1 patient both by population sequencing and by pyrosequencing (34%), and persisted as a minority species during the fourth off-treatment cycle (13%) (Fig. 1B). On resumption of therapy (during the fourth on-treatment cycle), M184V was not detected by either method. M184V has been observed by others as the major mutation that appears during repeated STIs,8-11 and drug resistance selection is most likely to occur during STIs, when few mutations are required to confer resistance.12 M184V has been reported in 1 study to emerge as a minority at different times during an STI in 14 of 25 subjects.9 By contrast, the K65R mutation was never detected at any time point, either by population sequencing or minority species assay (upper 95% confidence limit for risk of resistance per cycle = 4.1%). This may be because persisting intracellular Tenofovir diphosphate levels are insufficient to select for resistance. In addition, because zidovudine (AZT) is known to counteract the selection of K65R,13 there may be an impact of very low levels of intracellular zidovudine triphosphate (AZT-TP). On the other hand, the fitness cost to K65R14 may lead to a rapid disappearance after selection, particularly during treatment interruption, leading to an underascertainment of resistance even with sensitive assays. The HIV-1 subtypes in this study were A (n = 7), AE (n = 4) and D (n = 7), however, research findings have documented preferential emergence of the K65R mutation in subtype C-infected patients.15 In vitro studies have supported this view, due to the presence of polymorphisms at positions 64, 65, and 66 in RT of subtype C viruses.16 Interestingly, highly active antiretroviral therapy-experienced patients infected with subtype A virus showed a lower propensity to develop K65R compared with subtype B and C viruses, despite similar exposure to antiretroviral agents that select for this mutation,17 which could partly explain lack of detectable K65R in the STI patients infected with subtype A HIV-1 isolates. Whatever the mechanism, the inability to detect K65R in our study, even as a minority species during an STI, may help allay fears about widespread emergence of this mutation on stopping TDF-containing therapy. In conclusion, our preliminary data suggest a low risk of K65R and M184V emergence after treatment interruption on a combivir/TDF regimen. We caution that this does not mean that treatment interruption strategies are beneficial; indeed, the STI randomization in DART was stopped early on the basis of increased clinical progression in the STI group. ACKNOWLEDGMENT We would like to thank all the patients and staff from all the centers participating in the DART trial. Adele L. McCormick, PhD* Ruth L. Goodall, PhD† Aengus Joyce, BSc* Nicaise Ndembi, PhD‡ Mike Chirara, PhD§ Pauline Katundu∥ Sarah Walker, PhD† David Yirrell, PhD¶ Charles F. Gilks, DPhil, FRCP# Deenan Pillay, MD, PhD* on behalf of the DART Virology Group and Trial Team *Division of Infection and Immunity, University College London, London, United Kingdom †Medical Research Council Clinical Trials Unit, London, United Kingdom ‡Medical Research Council/UVRI Uganda Research Unit on AIDS, Entebbe, Uganda §University of Zimbabwe, Clinical Research Centre, Harare, Zimbabwe ∥Joint Clinical Research Centre, Kampala, Uganda ¶Ninewells Hospital and Medical School, Dundee, United Kingdom #Imperial College London, London, United Kingdom

Human immunodeficiency virus type 1 (HIV-1) is classified into different phylogenetic subtypes, with subtype C representing more than half of the novel infections globally. However, there are relatively few subtype C envelopes available for study. We amplified 18 unique env genes from 13 patients who were infected with subtype C HIV-1 in six African countries and in Scotland to create replication-competent viruses. These envelopes are phylogenetically diverse across the subtype C spectrum, and have on average more N-linked glycosylation sites and slightly longer variable loops than previously described C envelopes. We found that CCR3 coreceptor usage is less prevalent in subtype C than in subtype B viruses, and these envelopes have varied sensitivity to neutralization. The subtype C chimeric viruses generated in this study will be useful for evaluating the breadth of neutralizing antibodies and other entry inhibitors.

Background: Some HIV infected individuals remain asymptomatic for protracted periods of time in the absence of antiretroviral therapy (ART). Virological control, CD4 T cell loss and HIV-specific responses are some of the key interrelated determinants of HIV-1 disease progression. In this study, possible interactions between viral load, CD4 T cell slopes, host genetics and HIV-specific IFN-gamma responses were evaluated in chronically HIV-1-infected adults.Methodolology/Principal Findings: Multilevel regression modeling was used to stratify clade A or D HIV-infected individuals into disease progression groups based on CD4 T cell slopes. ELISpot assays were used to quantify the frequency and magnitude of HIV-induced IFN-gamma responses in 7 defined rapid progressors (RPs) and 14 defined slow progressors (SPs) at a single time point. HLA typing was performed using reference strand conformational analysis (RSCA). Although neither the breadth nor the magnitude of the proteome-wide HIV-specific IFN-gamma response correlated with viral load, slow disease progression was associated with over-representation of host immunogenetic protective HLA B* alleles (10 of 14 SPs compared to 0 of 7; p = 0.004, Fisher's Exact) especially B*57 and B*5801, multiclade Gag T-cell targeting (71%, 10 of 14 SPs compared to 14%, 1 of 7 RPs); p = 0.029, Fisher's Exact test and evident virological control (3.65 compared to 5.46 log10 copies/mL in SPs and RPs respectively); p < 0.001, unpaired student's t-testConclusions: These data are consistent with others that associated protection from HIV disease with inherent host HLA B allele-mediated ability to induce broader Gag T-cell targeting coupled with apparent virological control. These immunogenetic features of Gag-specific immune response which could influence disease progression may provide useful insight in future HIV vaccine design.

HIV-1 positive blood samples were collected between 1999 and 2005 from population groups most at risk of HIV infection in Bangladesh through the national surveillance, from clients of the Voluntary Counseling and Testing (VCT) Unit for HIV at ICDDR,B and a survey of HIV in patients with tuberculosis. Partial sequences of the gag gene were used for subtyping the HIV strains by nested PCR using selective primers. Of the 198 HIV strains tested, subtype C (41.4%) was the commonest strain identified. Phylogenetic analysis of Bangladeshi subtype C strains showed that they clustered in polyphyletic branches representing HIV strains from different parts of the world. Most of the strains from injecting drug users (IDU) clustered together and were similar to Indian strains. The VCT strains however were very heterogeneous and clustered with strains from India, Myanmar, Ethiopia and Zimbabwe. Data suggest that there have been few introductions into the IDU population where the epidemic is driven by indigenous transmission. On the other hand there have been many and regular introductions of subtype C viruses through migrant workers in the VCT group. Very little overlap was observed in the strains obtained from IDU and those from other population groups.

Objectives: To determine whether there are differences in coreceptor use in subjects infected with HIV-1 envelope subtypes A and D that could explain the differences in progression rates between these subtypes in a rural Ugandan cohort. Methods: HIV-1 was subtyped in env by V3 sequencing or heteroduplex mobility assay. Coreceptor use was determined by the ability of the isolates to replicate in U87 CD4+ cells expressing different coreceptors. The Fisher exact test was used to examine the relation between coreceptor use and subtype, clinical stage, and V3 charge. The Kruskall-Wallis nonparametric test was used to examine the association between median CD4 cell counts, coreceptor use, and subtype. Logistic regression was used to examine predicted coreceptor use at different CD4 groupings. Results: Isolates from 66 participants were analyzed. Thirty-one were infected with subtype A, and 35 were infected with subtype D. Although this work was based on a small sample size, we found statistically significant differences. The probability of having an X4 virus was higher in subtype D infections than in subtype A infections among those with a non-AIDS clinical status (Fisher exact test, P = 0.040). Logistic regression analysis, in which we predicted X4 use by subtype and stratified by CD4 group, confirmed these findings among those with a CD4 count >200 cells/μL (likelihood ratio test, P = 0.003). R5 viruses were associated with higher median CD4 cell counts than X4 or X4/R5 (Kruskall-Wallis test, P = 0.0045). A V3 charge of +5 and greater was highly associated with X4 virus (Fisher exact test, P = 0.006). Conclusions: These subtype differences in coreceptor use may partially explain the faster progression rates we have previously reported in individuals infected with subtype D compared with subtype A. Our observations may have implications for the future use of coreceptor inhibitors in this population.

SUMMARY The purpose of this study was to document the dynamics of HIV-1 subtypes in Scotland over a 6-year period. Viral RNA from all-new diagnoses was amplified by nested PCR and sequenced in the gag and/or env regions. Subtype was assigned by phylogenetic analysis, and aligned with demographic data including likely route and geographical origin of infection. We present data on 80% of all new diagnoses in Scotland between April 2000 and April 2006. Within the background of an expanding epidemic, subtype B predominates in men who have sex with men and intravenous drug users but there is a small but consistent number of UK-acquired infections in these risk groups caused by non-B subtypes. In heterosexuals, non-B subtypes acquired abroad, especially Africa, are still the largest group but again UK-acquired numbers are rising. The social and clinical significance of the spread of non-B subtypes in different ethnic and risk groups remains to be established.

Humoral responses against extra-cellular HIV-1 Tat may be beneficial as Tat has been implicated in the viral pathogenesis associated with HIV-1 disease progression. We determined the levels of anti-Tat IgG in sera of HIV-1 seropositive individuals from the Rural Clinical Cohort in Uganda using nine different Tat proteins representative of the major subtypes presently accounting for 97% of infections worldwide. We observed the presence of anti-Tat IgG able to react against the various subtypes tested, although none cross-reacted against all nine variants. We show that 46.25% of seropositive patients were able to recognise at least one Tat variant with 1:1000 sera dilution. We also show that the C terminus of Tat is the most variable region and an important epitope that might explain the limitation of cross-recognition of Tat antibodies regarding Tat variants. This study shows in seropositive patients that Tat can tolerate mutations without modification of its primary function but with changes in its immunogenic properties. These findings should be considered when designing Tat-based HIV-1 vaccines.

Although not linked to a disease, GB virus-C viraemia has been associated with an improved prognosis in HIV-1-co-infected individuals. Most studies have been conducted on men (men who have sex with men or injection drug users) infected with HIV-1 subtype B, whereas here we report on both male and female subjects from rural Uganda, predominantly infected via the heterosexual route with HIV-1 subtypes A and D. In a longitudinal study of 272 participants, 47 were GBV-C positive and 181 negative, as determined by reverse transcription-polymerase chain reaction, in both of two plasma samples taken a median of 5.0 years apart. The remainder either acquired (25) or cleared (19) infection. Multilevel regression analyses and Cox survival analyses revealed that participants chronically infected with GBV-C had a slower decline in CD4+ T cells (P < 0.001) and increased survival time (P=0.041) compared with GBV-C RNA-negative, HIV-positive adults. We show that the association between active GBV-C co-infection and improved survival of HIV-1-infected adults is not restricted to HIV subtype B, but is also observed in both males and females infected with HIV subtypes A and D.

The majority of studies of HIV-1 drug resistance have involved subtype B viruses. Here we have characterized subtype distribution and determined the levels of polymorphism at protease (PR) and reverse transcriptase (RT) drug resistance positions, in antiretroviral treatment-naive HIV-positive Ugandan patients. We have also investigated codon usage variability at these positions and assessed intersubtype recombination within the pol gene. The study population consisted of 187 patients, from a cohort established by the UK Medical Research Council Programme on AIDS in Uganda in 1990. Results indicate that 28.3% of patients were infected with subtype A (n = 53), 64.2% subtype D (n = 120), 6.4% A/D recombinant (n = 12), and 1.1% subtype C (n = 2). Variation in amino acid usage at drug resistance-associated positions was minimal between the two main subtypes (A and D) in RT, but there was appreciable variation in PR. Codon usage, however, was considerably more variable between subtypes A and D in both PR and RT. Thus, while no natural high-level resistance to antiretroviral therapy was detected in this cohort, subtypes A and D may possess different genetic barriers to be overcome in order to achieve resistance. With the increasing introduction of antiretroviral therapy into Africa, such information will be vital in our understanding and evaluation of the development of drug resistance as it occurs, and how to interpret resistance data the type of which has rarely previously been seen. This analysis also significantly increases the number of Ugandan PR and RT sequences characterized to date.